The development of fast and reliable DNA biosensors is of critical importance for the diagnostics/detection of infectious agents, for the identification of genetic mutations, for forensic investigations or food quality control, and will very likely continue to grow in the foreseeable future. Several sensitive approaches based on optical, electrochemical or magnetoresistive detection were reported over the years. Relatively few of these methods, however, offer the simultaneous advantages of simplicity, specificity, sensitivity and rapidity of detection without the use of chemical tagging of the DNA target or polymerase chain reaction (PCR) amplification.
A cationic polymeric transducer was previously reported to adopt distinct conformation when electrostatically bound to either ssDNA (single-stranded DNA) or dsDNA (double-stranded DNA) (U.S. Pat. No. 7,083,928). This technology allows optical detection of DNA material by fluorescence measurement in homogeneous medium, but is not as sensitive as desired (JACS 2004, 126, 4242-4244).
Further developments led to a combination of the cationic polythiophene with fluorophore-tagged ss-DNA probes to form a micellar system in which a Resonant Energy Transfer (RET) process leads to an amplification of the fluorescence signal emitted in the presence of target DNA material. This detection scheme, called “Fluorescence Chain Reaction” or FCR, allowed the optical detection by fluorescence of as few as 5 molecules of purified DNA from homogenous aqueous solution in only five minutes, and led to the first-ever demonstration of the direct detection of single nucleotide polymorphisms (SNPs) from clinical samples in such a short time, without the need for any nucleic acid amplification such as PCR (WO 2006/092063 A1, Leclerc et al.; JACS, 2005, 127, 12673-12676). However, the formation and evolution/conservation of micelles being dynamic phenomena, FCR relies on a particular arrangement of the fluorescent species within the self-assembled micelle-like aggregates which was shown to be strongly dependent on conditions of concentration, temperature and ionic strength in aqueous media (Langmuir, 2007, 23, 258-264). Therefore, improvements in the method to provide stabilization of the aggregates towards chemical and physical changes in their local environment and hence greater robustness are needed to ensure more reproducible analytical results.
It was recently shown that aggregates similar to those reported previously could be immobilized on a 2D solid support for DNA detection (international application NO.: PCT/CA2007/000857 published on Nov. 22, 2007 under No. WO2007/131354, Anal. Chem., 2006, 78, 7896-7899). The aggregates were covalently linked to the surface of a glass slide. Thus prepared, the slide displayed RET signal amplification (showing that the FCR behaviour of the aggregates was retained after the grafting process) and could be stored for extended periods in the dark and a dry atmosphere. After hybridizing with target DNA for 60 minutes, washing with a surfactant solution and water and finally drying the slides, the fluorescence signal was collected by a conventional microarray reader. However, whereas a molecular detection limit of 300 20-mer DNA target molecules was reported for 0.4-μL sample droplets, the volumetric detection limit reported for glass slide based FCR is significantly poorer than that reported for FCR detection in homogenous media (5×10−16 vs. 3×10−21 mole/L, respectively). Given that the majority of infectious diseases need to be diagnosed promptly in order to be curable and only a few pathogens are usually present in the blood or sputum (with sample volumes ranging from a few tens of μL to a few mL) at the onset of an infection, the volumetric detection sensitivity provided by glass slide based FCR is insufficient for PCR-free detection of such low levels of DNA material. Furthermore, detection of genomic DNA material (i.e. longer DNA chains typical of those found in clinical or biological samples), which is usually more difficult due to steric hindrance and rehybridization of the free overhanging tail of the capture DNA strand with its complementary strand (Peytavi et al, Biotechniques 2005), was not demonstrated with glass slide based FCR.
The poorer volumetric detection limit and longer hybridization time of glass slide FCR vs. homogenous FCR betray an inherent limitation of the 2D microarray-based format, i.e. the finite speed of diffusion of target molecules towards the immobile glass slide surface and the grafted aggregates is the key limiting factor when attempting to transfer FCR detection from homogeneous media to a static solid support while still retaining the former's detection speed and sensitivity. Because of this finite speed of diffusion, extending the application of this detection scheme to larger sample volumes would only result in poorer molecular detection limits. In other words, the procedure used to expose the sample to the grafted aggregates (deposition of individual microdroplets of sample over each grafted spot) cannot be extended to larger sample volumes without incurring signal losses due to incomplete analyte extraction from the sample.
Furthermore, the procedure used to graft the micelle-like aggregates onto the slides requires numerous chemical steps in different aqueous media as well as exposure of the grafted aggregates to the atmosphere and drying. Examples abound in the literature that underline the paramount importance of the experimental protocol used to deposit micelles of various types on slides for their microscopic examination, and in particular in the care needed when going from an aqueous to an organic and finally to a dry environment, in order to preserve their structural integrity and activity (Int. Dairy J. 2004, 14, 1025-1031). Since FCR signal amplification is known to hinge on a particular arrangement of fluorophores within the aggregates, which brings the fluorophore acceptors in close proximity to each other (Langmuir 2007, 23, 258-264; J. Fluoresc. 2006, 16, 259-265), it is thus desirable to transfer the aggregates in the native form obtained in solution onto a solid support in such a way that their photonics properties will not be altered. Therefore, the poorer detection sensitivity of slide based FCR vs. homogenous FCR could also be attributable in part to a modification in the conformation of the aggregates (form, size, density) caused by the drastic changes in the environment of the aggregates during their transfer from the homogenous solution to the slide surface.
Micrometer- or nanometer-sized particles or beads are commonly used for the detection of biomolecules. Most applications involve ss-DNA probes (labeled or unlabeled) that are covalently grafted on the beads (U.S. Pat. No. 6,544,746B2) These beads may then be manipulated or concentrated, usually by means such as magnetic fields or filtration. Capture of the targets by these probes is typically followed by the transduction of the “probe-target” recognition event (for example, using a minor groove intercalator for the double DNA helix, or a sandwich assay approach) (U.S. Pat. No. 5,821,066). However, since bead-based detection does not in itself procure an amplification of the optical signal, detection of ultralow levels of DNA material still requires prior amplification of the target sought to be detected
It was recently shown by Dubus et al. that DNA detection using the polythiophene transducer described previously was achievable directly on particles either using highly diluted suspension, i.e. homogeneous dispersion of the particles in solution or by confining particles in a small detection volume (Anal. Chem. 2006, 78:4457-4464). This approach, though sensitive and specific, does not meet the level of sensitivity reported for the technique known as FCR.
There thus remain a need to improve tools and methods for detection of nucleic acid and protein targets.
The present invention seeks to meet these needs and other needs.